A mouse monoclonal antibody against glycophorin A produced by in vitro stimulation with human red cell membranes.

Human erythrocyte membranes were used as antigen for production of mouse monoclonal antibodies against blood group related structures by in vitro immunization. Culture medium supernatant of PHA and PMA stimulated mouse thymus cells was used as source of cytokines. The selected antibody designated 124,D-7 (isotype IgM) was found to directly agglutinate all human red cells, except the rare erythrocytes En(a-) which lack glycophorin A. Immunoblotting showed faint bands in the positions of glycophorin A, whereas no binding occurred to glycophorin B. Inhibition of agglutination with purified glycophorin A and peptides suggests that the epitope is located within the amino acid residues 35-40. Rat and chicken erythrocytes also reacted with the antibody, whereas mouse erythrocytes were only agglutinated at very low dilutions of ascitic fluid.